A Modern Look at Protein Stability

Hazing or cloudiness in bottle is often thought of as a visual defect in wine and is considered a flaw to most consumers.  Once cause of this hazing is due to unstable heat proteins. The most well characterized trigger of unstable proteins becoming visible in wine is due to exposure to warm / hot bottle temperatures (~130°F); think of a bottled wine inadvertently left in a car on a hot summer day. This heat treatment causes the normally globular-shaped proteins to unfold and self-aggregate (chemically bind) with other unstable proteins, thus becoming visible and forming a haze over time (Fig. 1).

Similar to tartrate stability, winemakers often remove unstable proteins in bulk production before bottling. The textbook solution to remove these proteins in wine is to use Bentonite. Bentonite, a natural volcanic clay carries a negative charge in wine and is able to bind with unstable heat proteins. Through a simple bench trial, a winemaker can determine the exact amount of Bentonite needed to protein stabilize a wine.

Despite the effectiveness of this procedure, adding high doses of Bentonite can strip wine of positive organoleptic character, as is often the case in cold climate wine production. It is now well understood, that cold climate wines are rich in unstable proteins because they contain greater amounts of Pathogenisis-Related (PR) proteins, a characteristic inherited from their native Vitis sp. background.1

Alternative (Practical) Methods for Tackling Unstable Proteins

These same unstable proteins are also responsible for in the infamous tannin-binding mechanism, yielding a wine that is lower in tannin. Using this tannin-binding mechanism to our advantage, exogenous tannins added during fermentation (see sacrificial tannin theory) may also be used to diminish the amount of unstable protein. Tannins readily react and precipitate with proteins and this practice may be considered a first line defense at diminishing the concentration of unstable protein in wine.2 The timing, concentration, and the best tannin to use for this practice have yet to be defined.

More recently, in conjunction with Bentonite, an enzymatic means of improving protein stability in wine has been discovered. In short, Scottzyme KS (Kitchen Sink) is capable of disrupting the wine matrix so that less Bentonite is needed to protein stabilize wine.

The current theory is that the PR proteins are in a colloidal state (a complex interaction between proteins, polysaccharides, and polyphenols) and therefore not readily available to interact with Bentonite.  Adding KS prior to Bentonite addition, cleaves these colloids into more chemically reactive forms of unstable protein, requiring less Bentonite to protein stabilize (see Table 1).  As Scottzyme KS is both a pectinase and a protease used primarily to clarify wine, it is also possible that the side protease activity is directly decreasing the amount unstable protein (more on that below).

Regardless of the mode of action, in these bench trials wines were treated with 4# Bentonite per 1000 gallons of wine, 12-24 hours after Scottzyme KS addition (0.079 mL/L). The KS enzyme + Bentonite treated wines decreased unstable proteins by as much as 82% relative to the untreated control (no KS, only Bentonite).  For most of the wines, addition of KS enzyme was enough to render the wine protein stable at the current concentration of Bentonite!   Furthermore, the greater the concentration of unstable the proteins (as indicated by higher NTU in the untreated control), the more effective the KS treatment was at decreasing unstable protein.   Further investigation with regards to other enzymes, concentration, and contact time is warranted but preliminary results seem promising. This treatment provides a practical means of decreasing the amount of Bentonite needed to protein stabilize a wine.

On the Horizon

Researchers have known for quite some time that for a specific wine variety, unstable proteins in wine are measured in different concentrations as a function of yeast strain. As researchers further characterized the cause behind these yeast strain differences, two components emerged as beneficial to increasing protein stability: mannoprotein and chitin concentration (both derived from the yeast cell wall).

  1. a) Mannoproteins are typically associated with increasing the mouthfeel of wine through lees contact during ageing. However, in this study mannoprotein additions were used as a fining agent. Result efficacy varied as function of mannose to glucose ratio (the higher the mannose ratio the better), leaving room for further optimizing in this strategy. Moreover, mannoprotein treatment did affect the organoleptic properties of the wine.3
  1. b) Similar to the Mannoprotein study, researchers also discovered that yeast cell wall Chitin concentration (a structural polysaccharide) can remove unstable proteins in concentration dependent manner. However, before recommending this treatment, the effect of chitin addition to wine’s organoleptic properties must be further assessed, as it is probable that chitin will react with other wine constituents.4

Lastly, researchers have been also focusing on finding a specific protease enzyme that can attack and degrade these unstable proteins. The challenge with this strategy is to find an enzyme that is active at low pH, will attack only this subclass of proteins, and renders positive protein stability results. The first product on the foreign market (Proctase) has proven effective but first requires flash pasteurization of the wine before enzyme treatment.5 Heating the wine first promotes protein denaturation and unfolding so that the protease can better attack these proteins. However, flash-pasteurization is likely to be a deal breaker in this process, as most wineries in the Midwest do not have access to such technology or cannot justify the expense. Further investigation of proteases from other yeasts is under way and some preliminary results have yielded positive results thus far, without the need for a heating pre-treatment. Long-range, if a suitable protease is discovered, this may be the best alternative to protein stabilizing wine without Bentonite.

Outro

Last but not least, the question I often get asked is do I really need to protein stabilize wine? More and more winemakers are in fact decreasing the amount of Bentonite used in the cellar, opting to increase the aromatic potential of the wine at the risk of potentially having more issues with protein stability (Note: the whole issue of what is “true” wine protein stability is another issue in itself).

In addition, is has recently been discovered that these same unstable proteins play an important role in the foam height and stability in sparkling wine production, begging the question if removal of these proteins should even be targeted in the first place.6 Regardless of this exception to the rule, as an industry, the one underlying theme is the transition away from Bentonite for protein stabilization. The solution likely lies in using alternative fining agents and/or specific protease enzymes.

Sources

  1. Springer and Sacks. 2014. JAFC 62.
  2. Enartis. February 2018. Total Wine Stability.
  3. https://www.sciencedirect.com/science/article/pii/S0308814614003525
  4. http://aem.asm.org/content/early/2018/04/23/AEM.00668-18.abstract
  5. Beyond Bentonite. AWRI. November / December 2012
  6. Scott Labs. 2017 Fermentation Handbook. Pg. 103